The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

Abstract

The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50–pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.

‘Come together’—The Regulatory Interaction of Herpesviral Nuclear Egress Proteins Comprises Both Essential and Accessory Functions

“…( A ) Chemical structure of the benzimidazole riboside MBV. ( B ) The mode of antiviral activity of MBV was determined by a quantitation of viral encapsidated genomes [ 128 ]. The virus-specific qPCR-based measurements were performed after cellular fragmentation, i.e., using nuclear versus cytoplasmic fractions, using the recently established nuclear egress assay [ 125 ].…”

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target

“…The herpesviral nuclear egress complex (NEC) represents a functionally highly important determinant of viral replication efficiency. Recent studies on HCMV indicated that the disruption of the hook-into-groove interaction of pUL50 and pUL53 inhibits the late phase of viral replication [ 24 , 34 , 40 , 46 ]. Considering the features of core NEC sequences and structures, we addressed the question of whether distinct cysteine residues might be accessible to the attack of inhibitory warhead compounds.…”

“…This serves as a central platform for the formation of a multicomponent NEC, including core NEC-associated regulatory factors and kinases that localize at the inner nuclear membrane (INM). The multicomponent NEC involves emerin, p32/gC1qR, protein kinase C (PKC), cyclin-dependent kinase 1 (CDK1), possibly also CDK2 and further CDKs, the viral kinase vCDK/pUL97 and the peptidylprolyl cis/trans isomerase Pin1 [ 16 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. In particular, these kinases are of major importance for nuclear egress through their site-specific phosphorylation of lamins.…”

“…This leads to the disruption of the nuclear lamina in a Pin1-dependent manner, ultimately allowing the capsids to reach the INM [ 26 ], and for further steps of maturation through the nuclear envelope and the cytoplasm [ 16 ]. The integration of regulatory protein kinases into the nuclear egress process, in particular, vCDK/pUL97, has led to the specific consideration of kinase inhibitors as putative blocking agents of viral nucleocytoplasmic release [ 24 , 25 , 27 , 28 ]. Recently, covalently binding kinase inhibitors, referred to as warhead compounds, have attracted the specific interest of researchers and have already entered the various levels of clinical applications [ 29 ].…”

“…Concerning novel targeting strategies of anti-HCMV drugs, our group and many other researchers have studied a variety of different options of direct-acting and host-directed antivirals [ 27 , 32 , 33 ]. Very recently, we focused on the NEC as a highly promising target, which is composed of viral core components (core NEC) and, additionally, NEC-associated host and virus proteins (multicomponent NEC) [ 8 , 24 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. A crucial step towards an improved level of discovery of NEC-directed small molecules was our experimental resolution of crystallization-based 3D structures of α-, β- and γ-herpesviral core NECs [ 41 , 42 , 43 ].…”